Novel Sunifiram-carbamate hybrids as potential dual acetylcholinesterase inhibitor and NMDAR co-agonist: simulation-guided analogue design and pharmacological screening

Abstract An efficient method for synthesising NMDAR co-agonist Sunifiram (DM235), in addition to Sunifram-carbamate and anthranilamide hybrids, has been developed in high yields via protecting group-free stepwise unsymmetric diacylation of piperazine using N-acylbenzotiazole. Compounds 3f, 3d, and 3i exhibited promising nootropic activity by enhancing acetylecholine (ACh) release in A549 cell line. Moreover, the carbamate hybrid 3f was found to exhibit higher in vitro potency than donepezil with IC50 = 18 ± 0.2 nM, 29.9 ± 0.15 nM for 3f and donepezil, respectively. 3f was also found to effectively inhibit AChE activity in rat brain (AChE = 1.266 ng/mL) compared to tacrine (AChE = 1.137 ng/ml). An assessment of the ADMET properties revealed that compounds 3f, 3d, and 3i are drug-like and can penetrate blood–brain barrier. Findings presented here showcase highly potential cholinergic agents, with expected partial agonist activity towards glycine binding pocket of NMDAR which could lead to development and optimisation of novel nootropic drugs.


Computational methodology, Systems retrieval, preparation and molecular dynamics of the targets ability to bind to the glycine binding pocket of NMDA receptors .
The X-ray crystal structure of the ligand binding domain (LBD) of NMDA receptor (PDB ID: K4FQ ) was retrieved from RSCB protein data bank and prepared for molecular docking [1][2][3] by removing all nonstandard residues including water using UCSF Chimera. [4] The missing residues were modelled using MODELLER. [5] Molegro Molecular Viewer [6] was then employed to resolve torsion discrepancies. The two dimensional structures of the synthesized analogues 3a-j were sketched using Marving Sketch software [7]. Universal Force Field incorporated into Avogadro 1.2.0 software [8] was then employed to optimise the energy on the 2D structures and to build their 3D structures. The molecular geometries of the compounds were optimised using the steepest descent algorithm and saved for molecular docking. The compounds were then docked into the glycine binding pocket of the binding domain of the NMDA receptor using Autodock vina [9]. The pharmacokinetics and physicochemical properties of the compounds were then evaluated using SwissADME. [10] The compound with the highest docking score, low toxicity, favourable ADMET properties and Sunifiram (3a) were then selected for molecular dynamics simulation. The simulation process was performed by employing the Graphical Processing Unit version of the Particle Mesh Ewald Molecular Dynamics (PMEMD) engine in AMBER18 package [11]. AMBER FF14SB protein forcefield and the integrated pdb4amber program were used to parameterise the protein and modify the protein respectively [12], whiles the selected compounds were parameterised using the ANTECHAMBER module [13] of the AMBER 18 package which as well created the atomic partial charges for the compounds. The tLeap module of the AMBER18 package was then used to combine the protein and ligands into their docked complexes, neutralise the complexes by adding Na + and Clcounter ions and solvate the neutralised complexes with Transferable Intermolecular Potential with 3 Points (TIP3P) water box size of 8Å. An initial partial minimisation for 2500 steps employing 500 kcal/mol Å restraint potential was performed for the selected systems. This was followed by a full minimisation without energy restraint for 5000 steps. Heating of the systems was then performed gradually for 50 ps from 0 k to 300 k in NTP ensemble using Langevin thermostat [14] and a harmonic restraint of 5 kcal/mol A2. Berendsen barostat was used to ensure constant atmospheric pressure at 1 bar whiles the systems were then equilibrated at 300k for 1000 ps without energy restraints. MD productions was then performed for 250ns with the SHAKE algorithm used to restrain all hydrogen bonds [15]. CPPTRAJ and PTRAJ modules [16] integrated into the AMBER18 package were then used to analyse the trajectories and coordinates generated from the production. The data were then plotted and analysed using Origin data tool [17]. Structural visualisation was performed using Discovery Studio. [18] Thermodynamics calculations.
The differential binding affinity and stability of the selected compounds as well as the free binding energy of the complexes were investigated. The Molecular Mechanics/Poisson-Boltzmann Area method [19] was applied for the investigation due to its efficiency and widely reported reliability [20].
The binding free energy of this approach is depicted as follows: Where ΔGbind is taken to be the sum of the gas phase and solvation energy terms less the entropy (TΔS) term Where Egas is the total of the AMBER force field internal energy terms. Eint (bond, angle and torsion), the covalent van der Waals (Evdw) and the non-bonded electrostatic energy component (Eele). The solvation energy is denoted by the equation: Gnon-polar = γSASA + b The polar solvation contribution is denoted as GPB and Gnon-polar represents the non-polar contribution energy and is computed from the solvent assessable surface area (SASA). Which is obtained by the use of 1.4A water probe radius. Per-residue decomposition analyses were also performed to estimate individual energy contribution of the residues of the substrate pocket to the affinity and stabilisation of the compounds.

Cell Based Assay
The following codes were used for target compounds during cell based assay. 8-After treatment of cells with the serial concentrations of the compound to be tested incubation is carried out for 48 h at 37ºC, then the plates are to be examined under the inverted microscope and proceed for the MTT assay.

MTT -Cytotoxicity assay protocol
The MTT method of monitoring in vitro cytotoxicity is well suited for use with multiwell plates. For best results, cells in the log phase of growth should be employed and final cell number should not exceed 106 cells/cm2. Each test should include a blank containing complete medium without cells.

In vitro acetyl Choline release assay
The relative amount of ACh released in response to the synthesized Targets 3a and 3c-j was determined in A549 lung adenocarcinoma cells by Song et al protocol as follow: 1. A549 cells were grown in 5 ml serum-free RPMI (SF-RPMI) to 90 % confluence in 60 mm cell culture dishes in a cell culture incubator set at 5 % CO2 and 37 °C 2. On the day of the assay, 100 μM neostigmine (a chemical inhibitor of AChE in cells) was added to each plate for four hours at 37 °C. The plate contained 3 ml of media.
3. Four hours after the addition of neostigmine, the relevant concentration of test compunds 3a and 3c-j (1/2 IC50) were added and the cells were incubated at 37 °C for 36 hrs.
4. The supernatant (medium) was collected and spun at 800 × g.

5.
The supernatants were frozen at -80 °C and then lyophilized. The lyophilizer was set to a pressure of 10 micron Hg or below that value. The samples were lyophilized overnight.
6. Subsequently, the lyophilizate was reconstituted with 500 μl autoclaved distilled water, snap frozen in liquid nitrogen and stored at -80 °C for analysis.